Project Summary Depot-medroxyprogesterone acetate (DMPA), a contraceptive, has been associated with increased female acquisition of HIV through unknown mechanisms. We hypothesize that DMPA use increases vaginal species diversity, changing the vaginal microbiota in ways that promote genital inflammation and female HIV acquisition. We will use previously-collected vaginal swab samples from a prospective cohort of 81 postpartum Kenyan women who initiated DMPA and a control cohort of 25 women using non-hormonal contraception. Collaborating with the Fredricks laboratory at Fred Hutchinson Cancer Research Center, where these laboratory methods were pioneered, we will determine longitudinal changes in the vaginal microbiome using broad-range PCR with high-throughput sequencing. We will use taxon-specific quantitative PCR to determine quantities of the 12 most common vaginal bacteria and track their change over time. We will then correlate vaginal microbiota changes with peak DMPA levels, and assess the impact of DMPA on specific populations of vaginal anaerobes linked to HIV acquisition. The work proposed here leverages existing specimens collected specifically to assess biological consequences of DMPA use, and will use extensive socio-demographic and clinical metadata to perform multivariate statistical analyses comparing dynamic changes in microbiota species, richness and diversity with appropriate adjustment for confounding factors. Should this research reveal significant DMPA-related microbiota changes, our results would be applicable to ongoing efforts to create safer multi-purpose HIV prevention technologies and efforts to improve vaginal health in sub-Saharan Africa.